Abstract
Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. The broadly-used GAL promoter system for engineered yeast (Saccharomyces cerevisiae) suffers from several drawbacks; specifically, unintended induction during laboratory development, and unintended repression in industrial production applications, which decreases overall production capacity. Eukaryotic synthetic circuits have not been well examined to address these problems. Here, we explore a modularised engineering method to deploy new genetic circuits applicable for expanding the control of GAL promoter-driven heterologous pathways in S. cerevisiae. Trans- and cis- modules, including eukaryotic trans-activating-and-repressing mechanisms, were characterised to provide new and better tools for circuit design. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucose-mediated repression on the GAL promoter during the bioprocess. This delivered a 44% increase in production of the terpenoid nerolidol, to 2.54 g L−1 in flask cultivation. These negative/positive transcriptional regulatory circuits expand global strategies of metabolic control to facilitate laboratory maintenance and for industry applications.
Highlights
Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering
Yeast strains for promoter characterisation were obtained by transformation of an enhanced yeast green fluorescent protein expression cassette under the control of promoter of interest via single-copy genome integration (Fig. 1a)
In this work, we started with characterisation of basic genetic circuits, including eukaryote-like trans-activating and trans-repressing modules, and deployed new mechanisms to regulate GAL promoters aiming for application in metabolic engineering
Summary
Temporal control of heterologous pathway expression is critical to achieve optimal efficiency in microbial metabolic engineering. A eukaryote-like tetracycline-mediated circuit that delivers stringent repression was engineered to minimise metabolic burden during strain development and maintenance. This was combined with a novel 37 °C induction circuit to relief glucosemediated repression on the GAL promoter during the bioprocess. The system has limitations: (i) unintended auto-induction during routine strain maintenance and development is problematic when induced pathways result in metabolic burden or cellular toxicity; (ii) in prolonged pulsefeeding high-cell-density glucose processes, Mig1-mediated repression on GAL promoters may disrupt expression patterns; (iii) an increase in promoter strength is highly desirable to improve expression of bottleneck enzymes. The expanded GAL system was validated for repression or upregulation of heterologous sesquiterpene production in yeast
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